Method and apparatus for the identification of microorganisms

ABSTRACT

A method useful in identifying microorganisms and an apparatus useful to identify microorganisms, which apparatus comprises a flat sheet of absorbent material, the material characterized by a plurality of separate test zones therein, each zone including a substrate and an indicator, the substrate and indicator of each of the zones so selected on the sheet material so as to provide, by the reaction or a nonreaction of the microorganism in question with the substrate in the zone, an indication of the identification of the microorganism.

United States Patent [191 Sbarra METHOD AND APPARATUS FOR THE IDENTIFICATION OF MICROORGANISMS [75] Inventor: Anthony J. Sbarra, Milton, Mass.

[73] Assignee: Richard P. Crowley, Wellesley Hills,

Mass. a part interest [22] Filed: May 17, 1972 [21] Appl. No.: 254,193

[52] US. Cl. 195/103.5 R, 195/127, 195/139 [51] Int. Cl Cl2k l/04 [58] Field of Search l95/l03.5 R, 127, 139

[56] References Cited UNITED STATES PATENTS 12/1968 Streitfeld l95/l03.5 R 10/1958 Scherr l95/l03.5 R

OTHER PUBLICATIONS Auxotab Catalog; 1971.

[451 Dec. 24, 1974 Primary Examiner-A. Louis Monaccll Assistant Examiner-Robert J. Warden Attorney, Agent, or Firm-Richard P. Crowley [57] ABSTRACT 12 Claims, 3 Drawing Figures Pmmwvw 3.856.628

I4 IMPREGNATE PAPER DISCWITH CARBOHYDR D COLO INDICATOR TO SEPAR ZONES V I 2. DRY IMPREGNATED PAPER 3. PLACE PAPER ONTO P SURFACE OF CULTU PLATE STREAKED WITH MICROORGANISM 4. INCUBATE PLATE WITH PA PER DISC 5.0BSE COLOR CHANGES IN E IDENTIFY MICRO- ANISM ON CULTURE PLATE 3 METHOD AND APPARATUS FOR THE IDENTIFICATION OF MICROORGANISMS BACKGROUND OF THE INVENTION In general, the identification of various gramnegative and gram-positive microorganisms by their biochemical reactions has become a common procedure. Such techniques have proven most useful in assigning a genus and species to an unknown microorganism. Its identification is arrived essentially by the ability or inability of the particular microorganism to attack a specific substrate, the substrate being incorporated into a nutrient medium. Typically, the culture medium would contain appropriate nutrients to encourage the growth of particular microorganisms, a specific substrate (i.e., carbohydrate) and a suitable indicator, so that after a desired incubation period, the specific biochemical activity of the sample containing the microorganism may be determined. This would generally take a period of 24 to 48 hours.

The identification of microorganisms is often detected based on their ability or inability to cause fermentation of a carbohydrate in the presence of an indicator. For example, the biochemical identification of the genus Salmonellae may be accomplished by employing a culture media containing a variety of different substrates, such as lactose, glucose, ornithine, lysine and phenylalanine. Microorganisms belonging to the Salmonella group would not be expected to attack phenylalanine or lactose. They would, however, be expected to attack lysine, ornithine and glucose. Thus, any organisms suspected as belonging to the genus Salmonellae would not attack either lactose or phenylalanine. It should be pointed out that identification of microorganisms as outlined above is attempted only on pure cultures. Thus, after primary isolation (usually after a 24-hour incubation period) on an appropriate nutrient medium (i.e., blood agar medium), isolated colonies will appear, and these are transferred to individual, specific identifying mediums as alluded to above.

During the past few years, many efforts have been directed toward the development of rapid bacteriological identification techniques, so that information concerning the identification of particular microorganisms; for example, in urine samples, throat swabs and the like, may be rapidly determined and proper preventive or corrective action taken. All of the presently available identification techniques are based, first, on isolating the microorganisms from infected patient tissue on a primary culture. Isolation of the microorganisms is typically accomplished by innoculating a primary culture containing a nutrient with the bacteriological sample material and incubating the innoculated primary culture for a period of time, generally 24 hours, after which the resulting isolated microorganism is removed and innoculated into a number of different identification systems. Such identification systems include a tubed media containing nutrient material and specific identifying substrates, such as carbohydrates and appropriate indicators. These different tubed media are innoculated with the primary isolate and then incubated at 37C for an additional 24-hour period. After this incubation period, the reactions in each of the tubed media are read and recorded. Identification of the organism can be made by comparing the reactions noted with thosepreviously established for each organism.

An additional method employed. to identify microorganisms, although not frequently used, involves the impregnation of the particular substrate and culture medium on a paper disc. An additional disc is impregnated with an indicator. The prepared disc is now placed in culture tubes, water is added, and the solution then innoculated with the microorganism in question. As previously, the tubes are incubated and read after a suitable incubation period. By employing a series of culture tubes, the identification of the microorganism in question can be determined. Such techniques as described, while useful in identifying microorganisms, require a number of tubes, a variety of individual obser vations of each tube and assessments of the reaction and nonreaction of the products involved, and a comparison of the reactions in order to arrive at a suitable identification of the microorganism, all of which involve time and the possibility of error. Accordingly, a method which provides for a rapid and simple identification of microorganisms in a single procedure would be most desirable.

SUMMARY OF THE INVENTION My invention relates to a simple, rapid error-free method and apparatus for identifying microorganisms. My method provides a single-step procedure or technique by which the reaction or non-reaction of a microorganism in question can be ascertained. My apparatus useful in my method comprises an absorbent sheet material characterized by a plurality of different zones, the zones selected to determine by the reaction or nonreaction of a substrate and indicator in each zone with the identification of the microorganism. My method and apparatus allows a person to determine a plurality of different biochemical reactions, all on a single sheet of material, of the microorganism in question at the same time from a single test procedure and on a single culture plate.

My method comprises impregnating an absorbent sheet material, such as paper in the form of a paper disc, with a substrate subject to chemical reaction or nonchemical reaction with a microorganism and an identification agent to detect the chemical reaction or nonchemical reaction between the substrate and the microorganism in question. For example, the substrate might be a carbohydrate subject to fermentation, and an indicator subject to color change upon fermentation of the carbohydrate. The sheet material is impregnated in a plurality of zones, each zone containing a different substrate and an indicator. The zones may be placed in any position or form, and generally there will be from four to eight zones in each paper disc, each zone separated from the other zone so that the chemical reaction in one zone will not be obscured, overlapping with the chemical reaction of another zone. impregnation of the paper disc may be accomplished by placing a drop of the substrate and identifying agent on the paper disc and drying the disc rapidly. If desired, each particular zone may be identified and numbered, such as by placing a printed circle line around each zone, and numbering the zone, providing a code substrate identifying agent in each zone, or the color change in each zone required to identify each particular microorganism.

My method comprises placing the impregnated sheet material containing the various zones selected for a particular microorganism onto the top surface of a culture plate on which the microorganism in question has been streaked out, the culture plate containing the streaked microorganism in question with the absorbent sheet material contained in the zones, and the top surface is then incubated, and after an appropriate incubation period of time, the culture plate is removed and observation made of the reaction occurring in the various zones of the absorbent sheet material. Thus, in a rapid, simple and effective manner, on a single sheet of paper and culture plate, the microorganism in question could be rapidly identified. Since the substrate and indicator would, by simple diffusion, diffuse into the medium, and by observing the color change on the absorbent sheet material about each zone, identification of the microorganism can be made. If desired, the absorbent sheet material may be dampened with a little water while being placed on the top surface of the culture plate in order to promote diffusion and rapid reaction. Of course, it is obvious that different multiple paper discs be prepared, each containing a different set of zones and different substrates and indicators, each paper disc selected to identify a particular microorganism. The different substrates and indicators to be used would be determined by the nature of the microorganism in question,

FIG. 1 is a plan view of an impregnated paper disc of my invention.

FIG. 2 is a cross-sectional view of my disc along lines 22 of FIG. 1 in a culture plate.

FIG. 3 is a schematic illustration of my method of identifying a microorganism in an inoculated culture plate.

DESCRIPTION OF THE INVENTION The biochemical identification of, for example, the genus Salmonellae is accomplished by preparing a generally circular paper disc, which disc has been impregnated to form six generally circular zones, each zone impregnated as follows:

A phenol red and lactose B phenol red and mannitol C phenol red and adonitol D phenol red and dulcitol E phenol red and glucose F phenol red and sorbitol A bacteriological sample isolated containing the microorganism in question is then streaked out onto the surface of a culture plate containing trypticase soy agar. The surface of the culture plate is streaked with the sample, using a bacteriological loop, so that the entire surface is contacted with the sample. The impregnated paper disc which is generally designed to fit within the culture plate is then placed on top of the streaked sample. The culture plate is then incubated for about 24 hours at a temperature of 37C. The culture plate is then removed, and the paper disc then observed for a color change about the peripheral edges of each of the zones. If the culture sample contains the microorganism Salmonella, then the substrates in zones B, D, E and F would be fermented after incubation, and fermentation of these zones is detected by a color change in the indicator about the pheripheral edges of the zone. In the present case, a yellow zone indicating fermentation would be seen around the edges of the zone containing the fermented compounds, while no color change would be observed for zones A and C.

FIG. 1 shows an impregnated paper disc 10 of my invention which comprises an absorbent circular paper sheet 12 having a diameter less than the culture plate or Petri disc in which it is to be used. The paper sheet 12 contains six zones as set forth above, with a circular line 18 about the zones, and each zone identified by the alphabetical letter shown.

FIG. 2 shows the use of the paper disc 10 (along the lines 22 of FIG. 1) in a circular culture plate 14 containing an inoculated culture medium 16. The culture plate and the disc subsequently are incubated and the color changes of each zone observed to identify the microorganism.

FIG. 3 is a schematic illustration of the method of using my paper disc l0,to identify a microorganism in a manner set forth above.

In a similar manner, the biochemical identification of Escherichia coli may be accomplished by employing a paper disc with zones as follows:

phenol red and lactose phenol red and glucose phenol red and mannitol phenol red and dulcitol phenol red and adonitol phenol red and inositol If the sample is E coli, then after incubation, a color change would be observed around the peripheral edges of the zones containing the fermented glucose, lactose, dulcitol and mannitol, while inositol and adonitol would not be fermented by E coli.

My method and apparatus has been described by impregnating a single sheet of paper with the substrate identifying agent; however, it is also recognized that each zone may also be represented by a separate paper disc or absorbent disc placed or adhered to a carrier sheet, rather than having a single absorbent sheet containing impregnated zones. The absorbent sheet material should be selected to have a capillary attraction so that there may be contact between the sample on the culture plate and at least the peripheral edges of each zone so that some diffusion between the substrate and identification agent and the sample will occur during the incubation period to provide a reaction by which the microorganism can be identified. To provide good contact, the impregnated paper disc should be placed down firmly onto the surface of the culture medium to promote diffusion and reaction.

What I claim is:

1. Means useful to identify a microorganism through a change in abosrbent by the reaction or nonreaction of the microorganism with a carbohydrate comprising:

a. an essentially flat sheet of absorbent material adapted to be placed onto the top surface of a culture medium containing a microorganism where identity is to be determined;

b. the absorbent material free of restriction on at least one surface thereof and having a plurality of distinct and separate test zones thereon in a sufficient number such that the microorganism can be identified;

0. each test zone containing absorbed thereon in combination a carbohydrate and a pH color indicator of phenol red, which indicator changes color about the periphery of the test zone on the fermentation of a carbohydrate by the microorganism; and

d. the indicator employed in each test zone being the said phenol red and the carbohydrate in each test zone a different carbohydrate, whereby the absorbent sheet may be placed on top of a culture me dium containing a microorganism in a culture plate, and the change in color and lack of color change about the periphery of the test zones used to identify the presence or absence of a particular microorganism.

2. The means of claim 1 wherein the absorbent mate rial comprises a paper sheet having from four to eight test zones thereon.

3. The means of claim 1 wherein the carbohydrates are selected from the group consisting of lactose, mannitol, adonitol, dulcitol, glucose, sorbitol and inositol.

4. The means of claim 1 wherein the absorbent material is a circular paper sheet adapted to fit within and cover substantially the entire surface of a culture medium in a culture plate.

5. The means of claim 1 wherein the test zones are generally circular, and a means of identification employed to identify the test zones or the color change in fermentation by the microorganism.

6. The means of claim 1 wherein the test zones comprise separate absorbent discs secured to the absorbent sheet material.

7. A method of identifying a microorganism in a culture medium, which method comprises:

a. placing into contacting engagement onto the top surface of a culture medium containing or suspected of containing the microorganism where identity is to be determined an essentially flat absorbent sheet material, the sheet material having a plurality of distinct and separate test zones thereon in a sufficient number such that the microorganism can be identified, each test zone containing a sub strate and a pH indicator which, on reaction between the microorganism in the culture medium and the various substrates in the test zones, causes a change in color of the indicator about the periphery of the test zones, or no change in color by nonreaction, the test zones all having the same pH indicator and different substrates;

b. incubating the culture medium and the sheet material with the test zones for a period of time to permit the reaction or nonreaction of the microorganism with the substrates in the test zones; and

c. observing the change in color, if any, about the periphery of each test zone as a means for identification of the microorganism.

8. The method of claim 7 wherein the substrate is a carbohydrate selected from the group consisting of lactose, mannitol, adonitol, dulcitol, glucose, sorbitol and inositol.

9. The method of claim 7 wherein the sheet material is an absrbent paper disc, and which includes wetting the surface of the sheet material with water prior to incubation.

10. The method of claim 7 wherein the substrate is selected from the group of lysine, ornithine and phenylalanine.

11. The method of claim 7 wherein the indicator is phenol red.

12. The method of claim 7 which includes:

a. securing separate absorbent discs as test zones to the absorbent sheet material; and

b. placing the absorbent discs into contacting engage ment onto the surface of a culture medium.

=l l= l l 

1. MEANS USEFUL TO IDENTIFY A MICROOGANISM THROUGH A CHANGE IN ABSORBENT BY THE REACTION OR NONREACTION OF THE MICROORGANISM WITH A CARBOHYDRATE COMPRISING: A. AN ESSENTIALLY FLAT SHEET OF ABSORBENT MATERIAL ADAPTED TO BE PLACE ONTO THE TOP SURFACE OF A CULTURE MEDIUM CNTAINING A MICROOGANISM WHERE IDENITY IS TO BE DETERMINED; B. THE ABSORBENT MATERIAL FREE OF RESTRICTION ON AT LEAST ONE SURFACE THEREOF AND HAVING A PLURALITY OF DISTINCT AND SEPARATE TEST ZONES THEREON IN A SUFFICIENT NUMBER SUCH THAT THE MICROORGANISM CAN BE IDENTIFIED; C. EACH TEST ZONE CONTAINING ABSORBED THEREON IN COMBINATION A CARBOHYDRATE AND A PH COLOR INDICATOR OF PHENOL RED, WHICH INDICATOR CHANGED COLOR ABOUT THE PERIPHERY OF THE TEST ZONE ON THE FERMENT A THE MICROORGANISM; AND D. THE INDICATOR EMPOLYED IN EACH TEST ZONE BEING THE SAID PHENOL RED AND THE CARBOHYDRATE IN EACH TEST ZONE A DIFFERENT CARBOHYDRATE, WHEREBY THE ABSORBENT SHEET MAY BE PLACED ON TOP OF A CULTURE MEDIUM CONTAINING A MICROORGANISM IN A CULTURE PLATE, AND THE CHANGE IN COLOR AMD LACK OF COLOR CHANGE ABOUT THE PERIPHERY OF THE TEST ZONES USED TO IDENTIFY THE PRESSURE OR ABSENCE OF A PARTICULAR MICROORGANISM.
 2. The means of claim 1 wherein the absorbent material comprises a paper sheet having from four to eight test zones thereon.
 3. The means of claim 1 wherein the carbohydrates are selected from the group consisting of lactose, mannitol, adonitol, dulcitol, glucose, sorbitol and inositol.
 4. The means of claim 1 wherein the absorbent material is a circular paper sheet adapted to fit within and cover substantially the entire surface of a culture medium in a culture plate.
 5. The means of claim 1 wherein the test zones are generally circular, and a means of identification employed to identify the test zones or the color change in fermentation by the microorganism.
 6. The means of claim 1 wherein the test zones comprise separate absorbent discs secured to the absorbent sheet material.
 7. A method of identifying a microorganism in a culture medium, which method comprises: a. placing into contacting engagement onto the top surface of a culture medium containing or suspected of containing the microorganism where identity is to be determined an essentially flat absorbent sheet material, the sheet material having a plurality of distinct and separate test zones thereon in a sufficient number such that the microorganism can be identified, each test zone containing a substrate and a pH indicaTor which, on reaction between the microorganism in the culture medium and the various substrates in the test zones, causes a change in color of the indicator about the periphery of the test zones, or no change in color by nonreaction, the test zones all having the same pH indicator and different substrates; b. incubating the culture medium and the sheet material with the test zones for a period of time to permit the reaction or nonreaction of the microorganism with the substrates in the test zones; and c. observing the change in color, if any, about the periphery of each test zone as a means for identification of the microorganism.
 8. The method of claim 7 wherein the substrate is a carbohydrate selected from the group consisting of lactose, mannitol, adonitol, dulcitol, glucose, sorbitol and inositol.
 9. The method of claim 7 wherein the sheet material is an absrbent paper disc, and which includes wetting the surface of the sheet material with water prior to incubation.
 10. The method of claim 7 wherein the substrate is selected from the group of lysine, ornithine and phenylalanine.
 11. The method of claim 7 wherein the indicator is phenol red.
 12. The method of claim 7 which includes: a. securing separate absorbent discs as test zones to the absorbent sheet material; and b. placing the absorbent discs into contacting engagement onto the surface of a culture medium. 